CN116139331A - Multifunctional wound repair dressing loaded with bioactive glass and preparation method thereof - Google Patents

Abstract

The invention discloses a multifunctional wound repair dressing loaded with bioactive glass and a preparation method thereof, wherein the preparation method of the dressing comprises the following steps: synthesizing a quaternary ammonium salt modified polymer material; synthesizing phenylboronic acid modified polymer materials; preparing mesoporous bioactive glass microspheres by adopting a sacrificial template sol-gel method; dressing is prepared by phenylboronate and electrostatic action dynamic bond. The dressing has self-healing, antibacterial and biological activities, can be well injected and adhered to the wound surface, can accelerate the high-quality healing of the diabetic wound surface, and has good wound surface repairing effect.

Description

Multifunctional wound repair dressing loaded with bioactive glass and preparation method thereof

technical field

The invention relates to the technical fields of biomedical materials, tissue engineering and regenerative medicine, in particular to a multifunctional wound repair dressing loaded with bioactive glass for diabetic ulcers and a preparation method thereof.

Background technique

Diabetic ulcers are one of the most serious complications of diabetes, with delayed healing and excessive inflammation and impaired extracellular matrix function. Diabetic foot ulcers have become the leading cause of amputations in many countries. The main factors affecting the healing of diabetic ulcers are vascular network damage and bacterial infection. Reconstruction of the vascular network and elimination of bacteria are crucial for the treatment of diabetic ulcers. In addition to controlling blood sugar in patients, the treatment of diabetic ulcers is often achieved with the help of some functional materials. However, most of the current clinical materials have the disadvantages of single function and low biological activity, and cannot effectively repair wounds.

Hydrogel can absorb tissue exudate, maintain wound moisture and exchange gas (O ₂ , CO _{2 ,} etc.) near the wound in time, and is the best material for wound repair. More importantly, the performance of hydrogels can be designed, and multifunctional hydrogels with antibacterial, self-healing, and adhesion properties can be designed according to actual clinical needs to improve their practicability. At the same time, hydrogel is also an excellent carrier for biologically active substances. Bioactive dressings can accelerate wound repair from endogenous channels. Growth factors are often used clinically to achieve biological activity, but their disadvantages of high price and short half-life limit their application. Mesoporous bioactive glasses (MBGs) are inorganic repair materials with high specific surface area, high bioactivity and compatibility, which can promote the formation of blood vessels, the deposition of extracellular matrix (ECM) and collagen fibers to accelerate wound healing. Therefore, the multifunctional wound repair dressing loaded with bioactive glass based on practicality provides an alternative material for the treatment of diabetic ulcers.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and propose a multifunctional wound repair dressing for diabetic ulcers loaded with bioactive glass and a preparation method thereof. The preparation has injectability, self-adaptability, self-healing property, adhesiveness, antibacterial property and biological activity, can be well injected and adhered to the wound surface, and has good wound repair effect.

In order to achieve the above object, the technical solution provided by the present invention is: a method for preparing a multifunctional wound repair dressing loaded with bioactive glass, comprising the following steps:

1) Use cetyltrimethylammonium bromide (CTAB) and ethyl acetate (EA) to form a microemulsion system, add alkaline solution to corrode the template and act as a system catalyst, then add tetraethyl orthosilicate, triphosphate Mesoporous bioactive glass microspheres were prepared by sol-gel reaction of ethyl ester and calcium nitrate tetrahydrate;

Dissolve the quaternary ammonium salt modified polymer material in PBS to obtain A solution, dissolve the phenylboronic acid modified polymer material in PBS to obtain B solution; wherein, the quaternary ammonium salt modified polymer material and phenylboronic acid modified Polymer materials are dissolved in PBS at different concentrations;

2) Add the prepared mesoporous bioactive glass microspheres to A solution or B solution at a specific concentration and stir evenly to obtain C solution, and then mix A solution or B solution without mesoporous bioactive glass microspheres with C solution is mixed, and a self-healing, antibacterial and bioactive hydrogel can be quickly prepared as a wound repair dressing. Finally, stirring is continued at room temperature for a preset time to ensure the stability of the generated hydrogel.

Preferably, in step 1), the alkaline solution is ammonia solution, sodium hydroxide solution or dodecylamine solution; the added amount of calcium nitrate tetrahydrate is 2-20g.

Preferably, in step 1), the quaternary ammonium salt-modified polymer material is one or a combination of two or more of gelatin, chitosan, collagen, and carboxymethyl chitosan.

Preferably, in step 1), the phenylboronic acid-modified polymer material is one or a combination of two or more of sodium alginate, chitosan, gelatin, dextran, and hyaluronic acid.

Preferably, in step 1), the concentration of the quaternary ammonium salt modified polymer material is 1-5 wt%, and the concentration of the phenylboronic acid modified polymer material is 1-5 wt%.

Preferably, in step 2), the added amount of the mesoporous bioactive glass microspheres is 0.2-2wt%.

Preferably, in step 1), the preparation method of the mesoporous bioactive glass microspheres is as follows: 0.5-5mL CTAB is dissolved in deionized water, and after CTAB is completely dissolved, 5-50mL of EA is added to form a microemulsion system; After the microemulsion system is stable, add 5-50mL of alkali solution dropwise; after stirring for the preset time, add 5-20mL tetraethyl orthosilicate (TEOS) dropwise and continue stirring for the preset time; then, add 0.5-5mL triethyl phosphate (TEP), 2-20g calcium nitrate tetrahydrate (CN); then stir at the set temperature for 2-12h; Centrifugal washing with water to obtain a crude product; finally, sintering the crude product in air at 500-800°C for 2-8 hours to remove CTAB, EA, ethanol and reaction monomers to obtain the final product, which is mesoporous bioactive glass microspheres.

Preferably, in step 1), the preparation method of the quaternary ammonium salt-modified polymer material is as follows: dissolve the polymer material in an aqueous solution of acetic acid at a concentration of 2-10 wt%, and dissolve it completely at 25-55°C, A mixture is obtained; then, glycidyl trimethyl ammonium chloride GTMAC is added to the above mixture, wherein it is added according to the ratio of glycidyl trimethyl ammonium chloride GTMAC to the amino group on the polymer material 1:1-5:1 , after the addition, stir and react at 25-55°C for 10-24h; then centrifuge the reaction product to remove unreacted substances; finally, obtain the quaternary ammonium salt modified polymer material by freeze-drying after deionized water dialysis.

Preferably, in step 1), the preparation method of the phenylboronic acid modified polymer material is as follows: the polymer material is dissolved in 2-(N-morpholine) ethanesulfonic acid (MES) solution to obtain a concentration of 0.5- 2wt% solution, adding carboxyl activator to activate for 20-60 minutes, then adding aminophenylboronic acid, stirring continuously for 12-48h under dark conditions, dialyzing the reaction solution in deionized water and freeze-drying to obtain phenylboronic acid modified polymer material.

The present invention also provides a multifunctional wound repairing dressing loaded with bioactive glass prepared by the above method, which is used for wound healing of diabetic ulcers. The dressing has self-healing, antibacterial and biological activities, and can well Injected and adhered to the wound, it can accelerate the high-quality healing of diabetic wounds and has a good wound repair effect.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The present invention combines multifunctional hydrogel with bioactive glass, which not only provides a moist healing environment for wound healing, but also has excellent antibacterial, blood vessel promoting and healing promoting functions, effectively solving the problem of complex pathological environment of diabetic ulcers. problem and promote high-quality healing of diabetic wounds.

2. The introduction of bioactive glass in the present invention can promote the expression of vascularization-related factors, effectively solving the problems of high price and low utilization caused by direct use of growth factors.

3. The present invention modifies chitosan with quaternary ammonium salt, which not only solves the problem of poor water solubility of chitosan, but also improves the antibacterial performance of chitosan.

4. The raw materials of the hydrogel prepared by the present invention are all natural high molecular polymers, without using chemical cross-linking reagents, and have good biocompatibility.

5. The hydrogel prepared by the present invention is prepared by electrostatic interaction and phenylboronate bond, has certain mechanical strength, and has excellent injectability, making it suitable for various irregular wounds. At the same time, the good self-healing and adhesion properties enable it to be applied to joint wounds, effectively avoiding the failure caused by the dressing falling off and breaking during use.

Description of drawings

Fig. 1 is a SEM image of the mesoporous bioactive glass of the present invention in Example 2.

Fig. 2 is the physical picture of the injectability verification of the hydrogel obtained in Example 2.

FIG. 3 is a verification diagram of the adhesion of the hydrogel obtained in Example 2. FIG.

FIG. 4 is a verification diagram of the self-healing property of the hydrogel obtained in Example 2.

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

The preparation of quaternary ammonium salt modified carboxymethyl chitosan used below comprises the following steps:

Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid at a concentration of 3 wt%, and dissolve completely at 40°C for 30 min. Then GTMAC was added to the above mixture according to the ratio of glycidyl trimethyl ammonium chloride (GTMAC) to the amino group of carboxymethyl chitosan at 1:1, and the reaction was stirred at 55° C. for 10 h. Then the reaction product was centrifuged at 4500 rpm for 30 min to remove unreacted substances. The supernatant was poured into pre-cooled acetone to obtain the crude product. The crude product was redissolved in deionized water and then dialyzed against deionized water for 7d. Finally, quaternary ammonium salt modified carboxymethyl chitosan was obtained by freeze-drying.

The preparation of phenylboronic acid modified gelatin used below comprises the following steps:

Get 5.33g 2-(N-morpholine) ethanesulfonic acid (MES) in 500mL deionized water, obtain the MES buffer solution that concentration is 0.05mol/L; Dissolve 5g gelatin in the MES buffer solution of 500mL and obtain concentration is 1wt % solution, adding a carboxyl activator to activate for 30 minutes, then adding an appropriate amount of aminophenylboronic acid, stirring continuously for 48 hours under dark conditions, dialyzing the reaction solution in deionized water for 3 days, and then freeze-drying to obtain phenylboronic acid-modified gelatin.

The preparation of the quaternary ammonium salt modified collagen used below comprises the following steps:

The collagen was dissolved in an aqueous solution of acetic acid at a concentration of 10 wt%, and completely dissolved at 25° C. for 30 min. Then GTMAC was added to the above mixture according to the ratio of glycidyltrimethylammonium chloride (GTMAC) to the amino group of collagen at 3:1, and the mixture was stirred and reacted at 55° C. for 24 h. Then the reaction product was centrifuged at 4500 rpm for 30 min to remove unreacted substances. Then dialyzed in deionized water for 7 days. Finally, quaternary ammonium salt modified collagen was obtained by freeze-drying.

The preparation of phenylboronic acid modified sodium alginate used below comprises the following steps:

Take 5.33g of 2-(N-morpholine)ethanesulfonic acid (MES) in 500mL of deionized water to obtain a MES buffer solution with a concentration of 0.05mol/L. Dissolve 4g of sodium alginate in 200mL of MES buffer solution to obtain a solution with a concentration of 2wt%, add a carboxyl activator to activate it for 60 minutes, then add an appropriate amount of aminophenylboronic acid, and continue stirring for 12 hours under dark conditions. Dialyzed in deionized water for 3 days and then freeze-dried to obtain phenylboronic acid-modified sodium alginate.

The preparation of quaternary ammonium salt modified chitosan used below comprises the following steps:

Dissolve chitosan in an aqueous solution of acetic acid at a concentration of 2 wt%, and dissolve completely at 55° C. for 30 min. Then add GTMAC to the above mixture according to glycidyl trimethylammonium chloride (GTMAC) and chitosan amino group 5:1, and stir and react at 55° C. for 15 h. Then the reaction product was centrifuged at 4500 rpm for 30 min to remove unreacted substances. The supernatant was poured into pre-cooled acetone to obtain the crude product. The crude product was redissolved in deionized water and then dialyzed against deionized water for 7d. Finally, quaternary ammonium salt modified chitosan was obtained by freeze-drying.

The preparation of phenylboronic acid modified hyaluronic acid used below comprises the following steps:

Take 5.33g of 2-(N-morpholine)ethanesulfonic acid (MES) in 500mL of deionized water to obtain a MES buffer solution with a concentration of 0.05mol/L. Dissolve 1 g of hyaluronic acid in 200 mL of MES buffer solution to obtain a solution with a concentration of 0.5 wt%, add a carboxyl activator to activate it for 20 minutes, then add an appropriate amount of aminophenylboronic acid, and continue stirring for 24 hours under light-shielding conditions. Dialyzed in deionized water for 3 days and then freeze-dried to obtain phenylboronic acid-modified hyaluronic acid.

Example 1

1. Dissolve 2mL CTAB in 100mL deionized water. After CTAB was completely dissolved, 30mL of EA was added to form a microemulsion system. After the microemulsion system was stable, 25 mL of dodecylamine solution (2 mol/L) was added dropwise. After stirring for 15 min, 10 mL of tetraethyl orthosilicate (TEOS) was added dropwise and continued stirring for 30 min. Then, 1 mL of triethyl phosphate (TEP) and 8 g of calcium nitrate tetrahydrate (CN) were sequentially added every 30 min. It was then stirred at 40 °C for an additional 4 h. After standing and aging for 12 hours, it was centrifuged and washed three times with absolute ethanol and deionized water to obtain a crude product. Finally, the crude product was sintered in air at 500°C for 2 hours to remove CTAB, EA, ethanol, reactive monomers, etc., to obtain mesoporous bioactive glass microspheres.

2. Add 20mg of quaternary ammonium salt-modified carboxymethyl chitosan and 50mg of phenylboronic acid-modified gelatin to 1mL PBS respectively, then stir at room temperature for 24h to obtain a concentration of 2wt% quaternary ammonium salt-modified carboxymethyl chitosan Sugar and 5 wt% phenylboronic acid modified gelatin solution.

3. Disperse 20 mg of mesoporous bioactive glass microspheres in 1 mL of phenylboronic acid-modified gelatin solution, then quickly add quaternary ammonium salt-modified carboxymethyl chitosan to the above system, and stir at room temperature for 30 minutes to obtain stable The hydrogel is used as a wound repair dressing, which can be effectively used for high-quality healing of diabetic wounds in diabetic ulcers.

Example 2

1. Dissolve 0.5mL CTAB in 50mL deionized water. After CTAB was completely dissolved, 5 mL of EA was added to form a microemulsion system. After the microemulsion system was stable, 5 mL of ammonia solution (2 mol/L) was added dropwise. After stirring for 15 min, 5 mL of tetraethyl orthosilicate (TEOS) was added dropwise and continued stirring for 30 min. Then, 0.5 mL of triethyl phosphate (TEP) and 2 g of calcium nitrate tetrahydrate (CN) were sequentially added every 30 min. It was then stirred for another 12 h at 40 °C. After standing and aging for 24 hours, it was centrifuged and washed three times with absolute ethanol and deionized water to obtain a crude product. Finally, the crude product was sintered in air at 800°C for 8 hours to remove CTAB, EA, ethanol, and reactive monomers, etc., to obtain mesoporous bioactive glass microspheres.

2. Add 50mg of quaternary ammonium salt-modified collagen and 10mg of phenylboronic acid-modified hyaluronic acid into 1mL PBS respectively, and then stir at room temperature for 24h to obtain a concentration of 5wt% quaternary ammonium salt-modified collagen and 1wt% phenylboronic acid-modified Hyaluronic acid solution.

3. Disperse 5 mg of mesoporous bioactive glass microspheres in 1 mL of phenylboronic acid-modified hyaluronic acid solution, then quickly add quaternary ammonium salt-modified collagen to the above system, and stir at room temperature for 30 minutes to obtain a stable hydrogel Glue is used as a wound repair dressing and can be effectively used for high-quality healing of diabetic ulcers in diabetic wounds.

The morphology of the mesoporous bioactive glass microspheres in this example is shown in Figure 1. The preparation of the mesoporous bioactive glass microspheres firstly forms a microemulsion system with CTAB and EA as a template, and then adds an alkali solution to promote the hydrolysis of EA. , corrode the microemulsion structure. After TEOS and TEP were added, the sol-gel reaction proceeded gradually from the outside to the inside as the alkali solution corroded the template, and then bioactive glass microspheres with mesoporous structure were obtained. SEM showed that the bioactive glass microspheres had a regular spherical structure and obvious pore structure on the surface, with a uniform particle size of about 300nm.

The injectable performance of the hydrogel in this example is shown in Figure 2. The network structure of the hydrogel is composed of phenyl borate and electrostatic interaction. Phenyl boron ester is a reversible dynamic chemical bond, and electrostatic interaction is a kind of Reversible physical forces have the ability to break and generate quickly. Therefore, the hydrogel will break when subjected to a strong shear force, and can quickly recover its force after the shear force is removed. Macroscopically, the hydrogel can be injected from a 16G needle and can be rapidly molded, indicating that the hydrogel has good injectability.

The adhesion performance of the hydrogel in this example is shown in Figure 3. The structure of phenylboronic acid is similar to that of dopamine, which can form hydrogen bonds and other forces with skin tissue, thereby achieving good adhesion. It can be seen from Figure 3 that the hydrogel can adhere to the human finger and will not fall off as the finger is stretched, which confirms the good tissue adhesion of the hydrogel.

The self-healing performance of this embodiment is shown in Figure 4. When the dressing is applied to joint wounds, it often fails due to the breakage of the dressing due to movement. Therefore, a hydrogel dressing with self-healing performance is very necessary. The hydrogel is formed by dynamic phenylboronate and electrostatic interaction bonds. After being damaged by external effects, it can quickly combine when it is in contact again, so that the hydrogel can heal. Macroscopically, the two fractured hydrogels can quickly heal and form a whole gel after 30s of contact.

Example 3

1. Dissolve 5mL CTAB in a certain amount of deionized water. After CTAB was completely dissolved, 50mL of EA was added to form a microemulsion system. After the microemulsion system was stable, 50 mL of sodium hydroxide solution (2 mol/L) was added dropwise. After stirring for 15 min, 20 mL of tetraethyl orthosilicate (TEOS) was added dropwise and continued stirring for 30 min. Then, 5 mL of triethyl phosphate (TEP) and 20 g of calcium nitrate tetrahydrate (CN) were sequentially added every 30 min. It was then stirred for another 2 h at 40 °C. After standing and aging for 16 hours, it was centrifuged and washed three times with absolute ethanol and deionized water to obtain a crude product. Finally, the crude product was sintered in air at 650° C. for 3 h to remove CTAB, EA, ethanol, reactive monomers, etc., to obtain mesoporous bioactive glass microspheres.

2. Add 10mg of quaternary ammonium salt-modified chitosan and 50mg of phenylboronic acid-modified sodium alginate to 1mL PBS respectively, then stir at room temperature for 24h to obtain a concentration of 1wt% quaternary ammonium salt-modified chitosan and 5wt% Phenylboronic acid modified sodium alginate solution.

3. Disperse 2 mg of mesoporous bioactive glass microspheres in 1 mL of phenylboronic acid-modified sodium alginate solution, then quickly add quaternary ammonium salt-modified chitosan to the above system, and stir at room temperature for 30 minutes to obtain a stable The hydrogel is used as a wound repair dressing, which can be effectively used for high-quality healing of diabetic wounds in diabetic ulcers.

The embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those skilled in the art, other changes or changes in different forms can be made on the basis of the above-mentioned embodiments, and it is not necessary and impossible to exhaustively enumerate all the implementation manners here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (10)

1. The preparation method of the multifunctional wound repair dressing loaded with bioactive glass is characterized by comprising the following steps of:

1) Forming a microemulsion system by using cetyl trimethyl ammonium bromide CTAB and ethyl acetate EA, adding an alkali solution to corrode a template and serve as a system catalyst, and then sequentially adding tetraethyl orthosilicate, triethyl phosphate and calcium nitrate tetrahydrate to perform sol-gel reaction to prepare mesoporous bioactive glass microspheres;

dissolving a quaternary ammonium salt modified polymer material in PBS to obtain a solution A, and dissolving a phenylboronic acid modified polymer material in PBS to obtain a solution B; wherein the quaternary ammonium salt modified polymer material and the phenylboronic acid modified polymer material are dissolved in PBS at different concentrations;

2) Adding the prepared mesoporous bioactive glass microspheres into the solution A or the solution B at a specific concentration and stirring uniformly to obtain a solution C, then mixing the solution A or the solution B without the mesoporous bioactive glass microspheres with the solution C to quickly prepare the hydrogel with self-healing property, antibacterial property and bioactivity as a wound repair dressing, and finally, continuing stirring at room temperature for a preset time to ensure that the generated hydrogel is stable.

2. The method according to claim 1, wherein in step 1), the alkali solution is an aqueous ammonia solution, a sodium hydroxide solution or a dodecylamine solution; the addition amount of the calcium nitrate tetrahydrate is 2-20g.

3. The method according to claim 1, wherein in step 1), the quaternary ammonium salt modified polymer material is one or a combination of two or more of gelatin, chitosan, collagen, and carboxymethyl chitosan.

4. The method according to claim 1, wherein in step 1), the phenylboronic acid modified polymer material is one or a combination of two or more of sodium alginate, chitosan, gelatin, dextran, and hyaluronic acid.

5. The method according to claim 1, wherein in step 1), the concentration of the quaternary ammonium salt modified polymer material is 1 to 5wt% and the concentration of the phenylboronic acid modified polymer material is 1 to 5wt%.

6. The method according to claim 1, wherein in step 2), the mesoporous bioactive glass microspheres are added in an amount of 0.2-2wt%.

7. The method of claim 1, wherein in step 1), the mesoporous bioactive glass microspheres are prepared by: dissolving 0.5-5mL of CTAB in deionized water, and adding 5-50mL of EA after CTAB is completely dissolved to form a microemulsion system; after the microemulsion system is stable, dropwise adding 5-50mL of alkali solution; after stirring for preset time, dropwise adding 5-20mL of tetraethoxysilane, and continuously stirring for preset time; then, adding 0.5-5mL of triethyl phosphate and 2-20g of calcium nitrate tetrahydrate in sequence at intervals; stirring for 2-12h at the set temperature; standing and aging for 12-24 hours, and centrifugally washing with absolute ethyl alcohol and deionized water to obtain a crude product; finally, sintering the crude product in the air at 500-800 ℃ for 2-8 hours to remove CTAB, EA, ethanol and reaction monomers, thus obtaining the final product, namely the mesoporous bioactive glass microsphere.

8. The method of claim 1, wherein in step 1), the method of preparing the quaternary ammonium salt modified polymeric material comprises: dissolving polymer material in 2-10wt% concentration in acetic acid water solution, and completely dissolving at 25-55deg.C to obtain mixture; then adding the glycidyl trimethyl ammonium chloride GTMAC into the mixture, wherein the mixture is added according to the proportion of the glycidyl trimethyl ammonium chloride GTMAC to the amino groups on the polymer material of 1:1-5:1, and stirring the mixture at 25-55 ℃ for reaction for 10-24 hours; centrifuging the reaction product to remove unreacted substances; finally, the quaternary ammonium salt modified polymer material is obtained through freeze drying after deionized water dialysis.

9. The method of claim 1, wherein in step 1), the method of preparing the phenylboronic acid modified polymeric material is as follows: dissolving a polymer material in a 2- (N-morpholine) ethanesulfonic acid solution to obtain a solution with the concentration of 0.5-2wt%, adding a carboxyl activating agent for activating for 20-60 minutes, adding aminophenylboric acid, continuously stirring for 12-48 hours under the condition of avoiding light, dialyzing the reaction solution in deionized water, and freeze-drying to obtain the phenylboric acid modified polymer material.

10. The multifunctional wound repair dressing loaded with bioactive glass, which is prepared by the method of any one of claims 1-9, is used for wound healing of diabetic ulcers, has self-healing property, antibacterial property and bioactivity, can be well injected and adhered to a wound, can accelerate high-quality healing of the diabetic wounds, and has good wound repair effect.

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